Estrogenic compounds, process for their production and pharmaceutical uses thereof

ABSTRACT

The presently disclosed and claimed inventive concept(s) provides new estrogenic compounds of the general formula 
     
       
         
         
             
             
         
       
     
     and pharmaceutical compositions containing them. The compounds of the presently disclosed and claimed inventive concept(s) are useful, for example, in hormone replacement therapies (HRT/ERT), and as contraceptives, and estrogenic hormone therapies. Also provided is a process for synthesizing the compounds of the presently disclosed and claimed inventive concept(s).

CROSS-REFERENCE TO RELATED APPLICATIONS Incorporation by ReferenceStatement

This application is a continuation of U.S. Ser. No. 13/431,201, filedMar. 27, 2012; which is a continuation of U.S. Ser. No. 13/091,776,filed Apr. 21, 2011, now U.S. Pat. No. 8,158,684, issued Apr. 17, 2012;which is a continuation of U.S. Ser. No. 12/365,821, filed Feb. 4, 2009,now abandoned; which claims benefit under 35 U.S.C §119(e) of U.S.Provisional Application Ser. No. 61/026,029, filed on Feb. 4, 2008. Theentire contents of each of the above-referenced patents and patentapplications are expressly incorporated herein by reference.

FIELD OF THE INVENTION

The presently disclosed and claimed inventive concept(s) pertains to thefield of estrogenic compounds. More particularly, the presentlydisclosed and claimed inventive concept(s) pertains to estrogeniccompounds that do not readily form quinones in vivo, and topharmaceutical compositions and methods comprising an estrogeniccompound of the presently disclosed and claimed inventive concept(s), ora pharmaceutically acceptable salt, ester or solvate thereof.

BACKGROUND

The safety of long-term steroid usage by women for purposes of estrogenreplacement therapy (ERT) or oral contraception (OC) is currently underscrutiny. Epidemiological data can be hard to interpret due to changingpatterns of usage and drug formulations. However, based on recentreviews by the WHI and others [1-6], we can at least estimate themagnitude of the problem. Analysis of the increased risk factor forbreast cancer for OC users would be about 1.2±0.2, and for ERT usersabout 1.5±0.2. Using data from the USA, if the median age of OC users is25 years, the very low rate of cancer incidence in that age groupcombined with the low risk factor implies very few additional cases per100,000 women. Given the uncertainty in the risk data and the perceivedsocial benefit, this is generally considered to be a small butacceptable risk [for a different view of OC risk using higher riskfactors; however, see ref. 7]. For ERT users, their median age of ca. 50yr has a much higher cancer incidence which, when coupled with theincreased risk factor, suggests that there would be about 100-150additional cases per 100,000 women. This is a significant number ofcases, and after review of the epidemiological data, current medicalthinking in Canada and the U.S.A. is summarized in a set ofrecommendations given by the Canadian Task Force on Preventive HealthCare in May, 2004 [6]:

Recommendations

-   -   1) Given the balance of harms and benefits, the Canadian Task        Force on Preventive Health Care recommends against the use of        combined estrogen-progestin therapy and estrogen-only therapy        for the primary prevention of chronic diseases in menopausal        women (grade D recommendation);    -   2) For women who wish to alleviate menopausal symptoms using        hormone replacement therapy (HRT), a discussion between the        woman and her physician about the potential benefits and risks        of HRT is warranted.

These recommendations raised warning flags for patients and physiciansalike, due to the strong demand for hormone replacement coupled with thepuzzling risk/benefit analysis implicit in the statement. Of course, apreferable alternative to traditional drugs used as hormone supplementswould be a safer, non-carcinogenic compound.

Etiology of Breast Cancer:

The etiology of breast cancer is complex, with hormone-dependent andhormone-independent components [3]. It was originally thought that theonly relation between estrogens and cancer was through their ability tostimulate abnormal cell proliferation via estrogen-receptor mediatedprocesses [see ref. 8 and references therein]. However, as a result ofnew evidence on the relationship between estrogens and cancer the fieldis undergoing a “paradigm shift”. A new mechanism of interest, whichinvolves the formation of catechol estrogens as metabolites and theirsubsequent oxidation to carcinogenic quinones, is not yet consideredproven to be the dominant cause of breast cancer, but an increasingamount of evidence in its favor is appearing [8-17].

Quinone Formation and Carcinogenesis:

The naturally occurring estrogens estradiol and estrone have the classicsteroid structure containing the A, B, C and D-rings, where estradiol isshown in FIG. 1. The B, C and D rings are saturated, but the A-ring isan aromatic phenol. Phenols are easily metabolized in the liver andelsewhere by the enzyme cytochrome P450 hydroxylase [22]. This leads tohydroxyl substitution at the positions adjacent to the first hydroxylgroup (situated at position 3 in the A-ring), forming 2-OH estradiol and4-OH estradiol. These metabolites, termed the “catechol estrogens” [9]can be further metabolized by oxidizing substances present in the cell,e.g., peroxidase/P450 or tyrosinase/O₂ [23], or even in the presence ofoxygen, to give the 2,3-quinone and the 3,4-quinone [9-17].

Quinones in general are electrophilic compounds which have a tendency tobe tumor initiators and promoters, and several such mechanisms are known[22]. They can damage DNA by combining with nucleic acid bases thuscausing replication errors [22]. They can deplete essential cellularantioxidants such as glutathione and thiol-containing proteins,subjecting the cell to oxidative stress [22]. They can act directly asfree radical generators via reduction to the semiquinone form andsubsequent redox cycling, producing superoxide ion [24]. Differentquinones show differing amounts of cytotoxicity due to these competingmechanisms; some, such as the naphthoquinones and anthraquinones arehighly cytotoxic [22].

FIG. 1 shows the biological scheme of quinone formation, starting fromthe natural hormone 17β-estradiol (hereafter “estradiol”). Here thequinone formed involves only the A-ring, i.e., it is a benzoquinone. Inthe case of one of the conjugated equine estrogens present in the ERTdrug Premarin (currently the third most prescribed drug in the USA), thenaphthoquinone was formed and it was shown that hamsters treated withthe naphthoquinone for 9 months showed 100% tumor incidence [11,12].This led Bolton, Cavalieri and co-workers to the conclusion that“metabolism of estrogens to catechols and further oxidation to highlyreactive o-quinones could play a major role in induction of DNA damageleading to initiation of the carcinogenic process” [8, 13, 15-17]. Thisshort summary describes the catechol-estrogen hypothesis of the etiologyof breast cancer.

ERβ-Selective Agonists:

The recent discovery that estrogens bind similarly to the two receptorsubtypes, ERα and ERβ, and that these receptors have different tissuedistributions, has resulted in major efforts to develop ligands whichare selective agonists for either receptor [32,36 and 37]. Suchcompounds have considerable potential for the treatment of a number ofsymptoms and/or diseases associated with estrogen deficiency, includinghot flashes, osteoporosis and cardiovascular problems [37]. Tamoxifen,and raloxifene, although developed before the discovery of the ERβsubtype, are now classified as selective estrogen receptor modulators(SERMs) [32]. Thus, tamoxifen and raloxifene exhibit both estrogenic andanti-estrogenic activity depending on tissue type. The anti-estrogenicactivity of both compounds has been exploited to prevent re-occurrenceof ER-positive breast cancer and the prevention of breast cancer in highrisk women. Both drugs act as an agonist in the bone and thereby helpprevent osteoporosis [37]. Tamoxifen, but not raloxifene, also acts asan agonist in the uterus and leads to an increased risk of endometrialcancer. However, neither of these compounds relieves hot flashes, themost common menopausal symptom.

In their search for ERα and ERβ-selective agonists most research groupshave targeted non-steroidal families of compounds, inspired by thenatural product lead structure genistein. Considerable success has beenachieved in this area for structures illustrated, for example, by WAY202196 and ERB-041 [38 and 39]. These compounds show not only strongbinding but also excellent selectivity for ERβ vs. ERα; the bindingaffinity ERβ/ERα ratios for these structures are: genisten (41); WAY202196 (78); ERB-041 (226). However, the latter highly ERβ-selectivecompounds appear to be devoid of classical estrogenic activity. They donot promote the growth of estrogen-dependent MCF-7 breast cancer cells,but also do not relieve hot flashes or protect against osteoporosis. Thevalue of genistein for the treatment of hot flashes has not beenunambiguously established, although soy products (containing genistein)are commonly used for this purpose.

There remains a need for estrogenic compounds that avoid the problem ofquinone formation, while retaining hormonal activity. Compounds found tohave such activity will be useful, for example, in hormone replacementtherapy (HRT/ERT), in estrogenic hormone therapies and ascontraceptives.

This background information is provided for the purpose of making knowninformation believed by the applicant to be of possible relevance to thepresently disclosed and claimed inventive concept(s). No admission isnecessarily intended, nor should be construed, that any of the precedinginformation constitutes prior art against the presently disclosed andclaimed inventive concept(s).

BRIEF DESCRIPTION OF THE FIGURES

This patent or application file contains at least one drawing executedin color. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a schematic of the hydroxylation of estradiol to form thecatechol estrogens 2-OH estradiol and 4-OH estradiol, followed by their(auto)oxidation to form the corresponding 2,3- and 3,4-quinones.

FIG. 2 depicts ligand-receptor interaction for estradiol in 2D and 3Drepresentations. The important H-bond networks at positions 3 and 17 areshown.

FIG. 3 provides a schematic overview of drug design implementation usedin the presently disclosed and claimed inventive concept(s). 1) acomputer cluster used for computational analysis, 2) a model of estrogenreceptor showing test ligand (blue) inside first amino acid envelope, 3)correlation of RBA predicted vs. experimental from docking program, 4)synthesis of novel compounds to be tested for Relative Binding Affinity(RBA). Not shown: Additional tests for hormonal potency, acute andlong-term toxicity.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the presently disclosed and claimed inventiveconcept(s) belongs.

An object of the presently disclosed and claimed inventive concept(s) isto provide estrogenic compounds, a process for their production andpharmaceutical uses thereof. In accordance with an aspect of thepresently disclosed and claimed inventive concept(s), there is provideda compound of Formula I, or a pharmaceutically acceptable salt, ester orsolvate thereof,

where

R₁ is H, halogen or CH₃;

R₂ is H, halogen or CH₃;

R₄ is H, halogen or CH₃;

R₅ is H, halogen, CF₃, C₁-C₅ alkyl, CH₂OH, CH₂OAc, CH₂CH₂OH, CH₂CH₂OAc,CH₂-aryl, CH₂-heteroaryl, CH═CH₂, CH₂CH₂SCH₃, CH₂CH₂SC₂H₅, CH₂CH₂SCH₂Ar,CH₂CH₂SCH₂-heteroaryl, OH, OCH₃, OC₂H₅, OCH₂Ar, OCH₂-heteroaryl, OAc,SCH₃, SC₂H₅, SCH₂Ar, SCH₂-heteroaryl, SOCH₃, SOC₂H₅, SOCH₂Ar,SOCH₂-heteroaryl, SO₂CH₃, SO₂C₂H₅, SO₂CH₂Ar, SO₂CH₂-heteroaryl, CN, CHO,COCH₃, COC₂H₅, CO₂H, CO₂CH₃, CO₂C₂H₅, CO₂CH₂Ar, CO₂CH₂-heteroaryl,CONH₂, CON(CH₃)₂, CON(CH₂)₄, CON(CH₂)₅; NO₂

R₉ is absent, H or OH

R₁₇ is H or C₂H.

Preferably, the compound of Formula I has the following structure ofFormula Ia:

In accordance with another aspect of the presently disclosed and claimedinventive concept(s), there is provided a composition comprising acompound of Formula I, or a pharmaceutically acceptable salt, ester orsolvate thereof, and a pharmaceutically acceptable diluent or excipient.

In accordance with another aspect of the presently disclosed and claimedinventive concept(s), there is provided a method of hormone replacementtherapy comprising administering a compound of Formula I, or apharmaceutically acceptable salt, ester or solvate thereof, to a subjectin need thereof.

In accordance with another aspect of the presently disclosed and claimedinventive concept(s), there is provided a method of oral contraceptioncomprising administering a compound of Formula I, or a pharmaceuticallyacceptable salt, ester or solvate thereof, to a subject.

In accordance with another aspect of the presently disclosed and claimedinventive concept(s), there is provided a method of estrogenic hormonetherapy comprising administering a compound of Formula I, or apharmaceutically acceptable salt, ester or solvate thereof, to a subjectin need thereof.

In accordance with another aspect of the presently disclosed and claimedinventive concept(s), there is provided a process for synthesizing acompound of Formula I, comprising coupling an enantiomerically pureketone of Formula II with a lithium compound of Formula III,

where R and R₆ are independently H, alkyl or a protecting group and R₁,R₂, R₄, R₅ and R₁₇ have the meaning defined above in relation to FormulaI.

The presently disclosed and claimed inventive concept(s) providesestrogenic compounds that avoid or minimize the problem of quinoneformation, which is typically associated with compounds currently usedin ERT. The compounds of the presently disclosed and claimed inventiveconcept(s) have the structure of Formula I, or are pharmaceuticallyacceptable salts, esters or solvates thereof,

where

R₁ is H, halogen or CH₃;

R₂ is H, halogen or CH₃;

R₄ is H, halogen or CH₃;

R₅ is H, halogen, CF₃, alkyl, CH₂OH, CH₂OAc, CH₂CH₂OH, CH₂CH₂OAc,CH₂-aryl, CH₂-heteroaryl, CH═CH₂, CH₂CH₂SCH₃, CH₂CH₂SC₂H₅, CH₂CH₂SCH₂Ar,CH₂CH₂SCH₂-heteroaryl, OH, OCH₃, OC₂H₅, OCH₂Ar, OCH₂-heteroaryl, OAc,SCH₃, SC₂H₅, SCH₂Ar, SCH₂-heteroaryl, SOCH₃, SOC₂H₅, SOCH₂Ar,SOCH₂-heteroaryl, SO₂CH₃, SO₂C₂H₅, SO₂CH₂Ar, SO₂CH₂-heteroaryl, CN, CHO,COCH₃, COC₂H₅, CO₂H, CO₂CH₃, CO₂C₂H₅, CO₂CH₂Ar, CO₂CH₂-heteroaryl,CONH₂, CON(CH₃)₂, CON(CH₂)₄, CON(CH₂)₅; NO₂;

R₉ is absent, H or OH; and

R₁₇ is H or ethynyl (i.e., CCH).

As would be readily appreciated by a skilled worker, the solvate can bea hydrate.

It should also be noted that if the stereochemistry of a structure or aportion of a structure is not indicated with, for example, bold ordashed lines, the structure or the portion of the structure is to beinterpreted as encompassing all stereoisomers of it. Moreover, any atomshown in a drawing with unsatisfied valences is assumed to be attachedto enough hydrogen atoms to satisfy the valences. In addition, chemicalbonds depicted with one solid line parallel to one dashed line encompassboth single and double (e.g., aromatic) bonds, if valences permit.

As used herein, the term “alkyl” is used to refer to a straight orbranched chain hydrocarbon having from 1 to 5 carbon atoms. The term“alkyl” includes saturated hydrocarbons as well as alkenyl and alkynylmoieties.

As used herein, the term “aryl” is used to refer to an aromatichydrocarbon group containing 6 to 10 carbon atoms.

As used herein, the term “halogen” is used to refer to fluorine, achlorine, a bromine or an iodine. A preferred example of such a halogenis a fluorine or chlorine.

As used herein, the term “heteroaryl” is used to refer to a 5- or10-membered aromatic heterocyclic group containing one or moreheteroatoms selected from an oxygen atom, a nitrogen atom, and a sulfuratom.

Preferably, the compound of Formula I has the following structure andstereochemistry:

wherein, R₁, R₂, R₄, R₅, R₁₇ are as defined above, and R₉ is H or OH.

In accordance with a specific embodiment of the presently disclosed andclaimed inventive concept(s) there is provided a compound having astructure of Formula I, or a pharmaceutically acceptable salt, ester orsolvate thereof, wherein

R₁ is H, F or CH₃;

R₂ is H or F;

R₄ is H or F;

R₅ is H, F, Cl, CF₃, CH₃, C₂H₅, nC₃H₇, iC₃H₇, CH₂OH, CH₂OAc, CH₂CH₂OH,CH₂CH₂OAc, CH₂-aryl, CH₂-heteroaryl, CH═CH₂, CH₂CH₂SCH₃, CH₂CH₂SC₂H₅,CH₂CH₂SCH₂Ar, CH₂CH₂SCH₂-heteroaryl, OH, OCH₃, OC₂H₅, OCH₂Ar,OCH₂-heteroaryl, OAc, SCH₃, SC₂H₅, SCH₂Ar, SCH₂-heteroaryl, SOCH₃,SOC₂H₅, SOCH₂Ar, SOCH₂-heteroaryl, SO₂CH₃, SO₂C₂H₅, SO₂CH₂Ar,SO₂CH₂-heteroaryl, CN, CHO, COCH₃ COC₂H₅, CO₂H, CO₂CH₃, CO₂C₂H₅,CO₂CH₂Ar, CO₂CH₂-heteroaryl, CONH₂, CON(CH₃)₂, CON(CH₂)₄, CON(CH₂)₅;NO₂;

R₉ is H or OH; and

R₁₇ is H or ethynyl.

Preferably, R₅ is H, F, Cl or CH₃.

In designing the compounds of the presently disclosed and claimedinventive concept(s), a multi-step process was employed. First apreliminary screen was performed on a computer using a program thatfacilitates the visualization of ligand-receptor interface, where thereceptor in this case is an estrogen receptor. This initial pre-screenwas done to identify candidate compounds suitable for furthercomputational analysis. A second computer-implemented screen wasperformed using a docking algorithm that provides a predicted relativebinding affinity (RBA) for each compound. This second screen wasperformed to identify a series of high affinity lead compounds. Finally,the lead compounds were synthesized and tested as described below. Theoverall process is depicted in FIG. 3.

Computational Drug Design

Molecular Operating Environment computing platform [MOE, 2008] wasselected for drug design. This software is developed by the ChemicalComputing Group (CCG) in Montreal and is attracting increasedinternational usage. PC Spartan was used to calculate ligand solvationenergies [29]. A validation study was performed on a set of 25 ligandsknown to bind to human recombinant ERα [11-13, 30, 31]. First, acomputer cluster (FIG. 3-1) was set up to handle the extensivecomputational requirements associated with conformational freedom ofligands and receptor flexibility [25,26]. Modifications to existingsoftware were required to make such a study feasible, and a “shellmodel” of the receptor protein was created (FIG. 3-2) which made studyof ligand-receptor interactions much faster, e.g., a total CPU time ofabout 1 day per molecule. Techniques were also introduced to allow theprotein geometry to relax to accommodate the ligand and tested various“scoring functions” to rank ligand poses; the latter were compared tothe log of the experimental RBA. Optimization of the training set andscoring function was performed and produced a strong correlation (FIG.3-3), which allowed prediction of novel “agonists” that would obey thedesign criteria.

In addition to the above development of the docking algorithm, recentimprovements in visualization of the ligand-receptor interface were madeby Chemical Computing Corporation which helped in establishing thepreliminary screen for optimal ligand binding. Briefly, the H-bondingnetwork which is optimum for estradiol was assumed to be important; thisprovides H-bond anchors at receptor residues Glu353, Arg394 and His524(FIG. 2). As in estradiol a water molecule was retained in the receptorcavity, which should participate in the H-bonding (FIG. 2). To optimizethe H-bond network the O—O distance should be near 11 Å, as inestradiol. Next, using FIG. 2 to visualize the receptor cavity, theligand should not cross the boundary of the cavity excessively, or theprotein will be unable to adjust sufficiently to accommodate it.Finally, the solvation energy of the ligand (computed using PC Spartansoftware [28]) must not be too large since the ligand must bede-solvated when entering the receptor. Constraints on the liganddescribed above are similar to a “pharmacophore model” discussed by JohnKatzenellenbogen and coworkers [33] and used as a screening techniqueprior to their own syntheses.

Using the criteria above as a pre-screen requires only about 5 minutesand allows rejection of many otherwise apparently promising compounds.Molecules which passed the pre-screening tests were sent to the dockingprogram described above and an RBA for each compound was predicted forbinding to ERα. A series of predicted high-affinity lead compounds weresynthesized and tested as set out below.

Synthesis of Compounds

In accordance with another aspect of the presently disclosed and claimedinventive concept(s), there is provided a process for synthesizing acompound of Formula I, comprising coupling an enantiomerically pureketone of Formula II with a lithium compound of Formula III,

where R and R₆ are independently H, alkyl or a protecting group and R₁,R₂, R₄, R₅ and R₁₇ have the meaning defined above in relation to FormulaI. As described in more detail below, the lithium compound of FormulaIII can be prepared using known techniques, for example, by reacting thecorresponding brominated compound nBuLi. The product of the couplingreaction is a mixture of unsaturated hydroxyl isomers, which aresubsequently subjected to dehydrogenolysis and deprotection (asnecessary) and the resultant isomers are separated to yield the compoundof Formula I.

As would be readily appreciated by a worker skilled in the art,alternative synthetic methods can be used to prepare the compounds ofthe presently disclosed and claimed inventive concept(s).

In describing the compounds of the presently disclosed and claimedinventive concept(s), it is important to note that their numbering isbased on steroid numbering, as shown below.

Compound 1, the first target molecule, was synthesized and studied byradiolabel assay to determine binding activity. Relative to estradiol(set at 100%) the RBAs for ERα and ERβ were determined to be 1.5% and21.5%, respectively. This demonstrated that ring A can be successfullycoupled to rings CD and that the first such compound shows selectivebinding, favouring ERβ by a factor of 15. However, it was found not toblock formation of quinones.

Activity Testing

The compounds of the presently disclosed and claimed inventiveconcept(s) are estrogenic and do not readily metabolize to quinones invivo. Following synthesis, the compounds were tested to confirm theirproperties are as predicted by the computational analyses describedabove.

Tests for Relative Binding Affinity:

These tests are well described in the literature [31]. Briefly, aradiolabel assay is used where competition for the receptor binding siteis set up between radiolabeled estradiol and the ligand underinvestigation. Displacement of the radioactivity means that the ligandshows binding to the receptor, and this is quantified to give therelative binding affinity (RBA). Here strong binding is highlycorrelated with hormonal potency (unless antagonists are designeddeliberately, which was not the goal of the presently disclosed andclaimed inventive concept(s)).

mRNA Transcription Assay:

This assay, which is a measure of hormonal potency, is also given in theliterature and follows standard protocols [12]. For hormone assays anestrogen response element driven luciferase will be transfected intoCOS-7 ER+ cells, to test for transcriptional activation by testcompounds relative to estradiol. Compounds with estrogenic activitycause luciferase production, which is monitored by luminescencedetection using standard well-known techniques.

Quinone Related Toxicity Assay:

The toxicity of test compounds is determined by exposure of intacthepatocytes to the test compound. Toxicity levels are expressed as LC50values following 2 hour exposure.

Tests for Carcinogenicity:

The protocol used in Pratt's laboratory on studies of retinoic acid andits receptor [35] will be used to test the compounds of the presentlydisclosed and claimed inventive concept(s). MCF-10A cells will be used,which are immortalized human mammary epithelial cells devoid oftumorigenic activity. These cells will be subjected to parent drug ormetabolites at increasing concentrations for periods between 5 and 25days. Cells will then be trypsinized and replated in soft agar at theend of the treatment period and assessed for anchorage-independentcolony formation. Some cells will continue to be exposed to drug byinclusion of the appropriate concentration to determine tumor promoteractivity, while others will remain without. Colonies will be isolatedand expanded. Incidence of mutation will be determined using the HPRTgene as an indicator by growth in 6-thioguanine. Commonly upregulatedgenes, including c-myc, p65 Rel-A, survivin, p53(mut) and PIN 1, will beassessed by immunoblot and RT-PCR. Transforming quinones, i.e., 4-OHequilenin, will be compared with the novel compound and its metabolitesin the MCF-10A transformation assay.

To assess tumorigenicity, ca. 1×10⁶ cells from 20 different colonieswill be injected subcutaneously into 8 week-old female nude mice. Thesemice are sexually mature and produce normal levels of endogenousestrogen. Mice will be examined on a weekly basis for tumor formationover a period of 1 year. Tumors will be measured and weighed aftersacrifice and parameters including ER/PR status and mitotic indexassessed. The results will permit comparison of the inherent tumorigenicactivity of estradiol to that of the compounds of the presentlydisclosed and claimed inventive concept(s). To be pharmaceuticallyuseful, compounds should exhibit low or zero tumorigenicity.

Pharmaceutical Compositions and Uses Thereof

The compounds of the presently disclosed and claimed inventiveconcept(s) are useful as estrogenic compounds. One aspect of thepresently disclosed and claimed inventive concept(s) provides methodsfor treating or preventing disorders related to estrogen functioning.For example, the compounds of the presently disclosed and claimedinventive concept(s) can be administered as alternatives to currenthormone/estrogen replacement therapies (HRT/ERT), estrogenic hormonetherapies and oral contraceptives. Accordingly, one aspect of thepresently disclosed and claimed inventive concept(s) provides a methodof hormone replacement therapy comprising administering a compound ofFormula I, or a pharmaceutically acceptable salt, ester or solvatethereof, to a subject in need thereof. Another aspect of the presentlydisclosed and claimed inventive concept(s) provides a method of oralcontraception comprising administering a compound of Formula I, or apharmaceutically acceptable salt, ester or solvate thereof, to asubject.

In another example, the presently disclosed and claimed inventiveconcept(s) provides a method of estrogenic hormone therapy (EHT)comprising administering a compound of Formula I, or a pharmaceuticallyacceptable salt, ester or solvate thereof, to a subject in need thereof.EHT is a general term used to refer to a broad range of indicationsincluding, but not limited to, female hypogonadism, osteoporosis,castration, primary ovarian failure, amenorrhea, dysmenorrhea,oligomenorrhea, lactation suppression, growth attenuation, and some maleinfertility or prostate cancer treatments.

The term “subject” as used herein, refers to an animal, preferably amammal, most preferably a human, who has been, is or will be the objectof treatment, observation or experiment.

Administration of the compound of the presently disclosed and claimedinventive concept(s) can be carried out via any of the accepted modes ofadministration or agents for serving similar utilities. Thus,administration can be, for example, orally, nasally, parenterally,topically, transdermally, or rectally, sublingually, intramuscular,subcutaneously, or intravenously in the form of solid, semi-solid,lyophilized powder, or liquid dosage forms, such as for example,tablets, suppositories, pills, soft elastic and hard gelatin capsules,powders, solutions, suspensions, or aerosols, or the like, preferably inunit dosage forms suitable for simple administration of precise dosages.The compositions will include a conventional pharmaceutical carrier orexcipient and a compound of the presently disclosed and claimedinventive concept(s) as the/an active agent, and, in addition, mayinclude other medicinal agents, pharmaceutical agents, carriers,adjuvants, etc.

In accordance with specific embodiments of the presently disclosed andclaimed inventive concept(s), such compositions will take the form of acapsule, caplet or tablet and therefore optionally also contain adiluent, a disintegrant, a lubricant, and/or a binder.

Alternatively, a compound of the presently disclosed and claimedinventive concept(s) can be formulated into a suppository using, forexample, about 0.5% to about 50% active ingredient disposed in a carrierthat slowly dissolves within the body, e.g., polyoxyethylene glycols andpolyethylene glycols (PEG), e.g., PEG 1000 (96%) and PEG 4000 (4%), andpropylene glycol.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, etc., a compound of the presentlydisclosed and claimed inventive concept(s) (e.g., about 0.5% to about20%), and optional pharmaceutical adjuvants in a carrier, such as, butnot limited to, water, saline, aqueous dextrose, aqueous cyclodextrin,glycerol, ethanol or the like, to thereby form a solution or suspension.

If desired, a pharmaceutical composition of the presently disclosed andclaimed inventive concept(s) can also contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,antioxidants, and the like.

Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in this art; for example, see Remington'sPharmaceutical Sciences, 18th Ed., (Mack Publishing Company, Easton,Pa., 1990). The composition to be administered will, in any event,contain a therapeutically effective amount of a compound of thepresently disclosed and claimed inventive concept(s).

The therapeutically effective amount of a compound of the presentlydisclosed and claimed inventive concept(s) will vary depending upon avariety of factors including the age, body weight, general health, diet,mode and time of administration, rate of excretion, drug combination,the severity of the particular disease-states, and the host undergoingtherapy.

The above paragraphs all apply to use of the compounds for monotherapyapplications, i.e., where only one compound at a time is used. However,the RBAs of the developed compounds show a range of binding affinitiesranging from strongly ERα-selective to strongly ER-β selective. It canbe desirable to use a mixture of both types of compounds as acombination therapy.

To gain a better understanding of the presently disclosed and claimedinventive concept(s) described herein, the following examples are setforth. It should be understood that these examples are for illustrativepurposes only. Therefore, they should not limit the scope of thispresently disclosed and claimed inventive concept(s) in any way.

EXAMPLES Example 1 Synthesis of ACD Estrogenic Compounds

Scheme 1 outlines a general scheme useful for the synthesis of the ACDring-containing compounds of the presently disclosed and claimedinventive concept(s).

The CD ring moiety, compound 3, was synthesized in enantiomerically pureform following an established literature procedure.

The A ring part, compound 1, was either obtained from a commercialsource as the corresponding phenol or was synthesized via knownliterature methods that typically involved purchasing the non-brominatedprecursor and then carrying out an electrophilic bromination usingeither Br₂ or N-bromosuccinimide in a suitable solvent.

The phenolic OH and the secondary OH in the D ring were protected (to—OR or —OR₆, respectively) in order to be non-reactive to then-Butyllithium (BuLi) treatment by conversion to ethers using groupssuch as CH₃—, PhCH₂—CH₂═CH—CH₂, CH₃OCH₂— [MOM], or tetrahydropyranyl,[THP] or conversion to silyl ethers such as tertbutyldimethylsilyl(tBDMS). Subsequent deprotection was performed using known methods fordeprotection. R and R₆ can be the same or different protecting groups.

Alternatively, the phenolic OH is not protected and the reaction wasperformed using an extra equivalent of nBuLi instead.

Furthermore, if the substituents on the A ring are reactive to nBuLi,then it can be necessary to include additional steps for protection (andsubsequent deprotection) of these substituents. Again, the protectionand deprotection steps were performed using standard methods.

Commercial nBuLi in tetrahydrofuran (THF) was used most commonly toconvert compound 2 into the nucleophilic lithiated derivative.Alternatively the magnesium derivative can be prepared using either Mgin ether or THF or isopropyl magnesium chloride in THF as the reagent tocarry out the bromine to metal exchange. The halogen metal exchangereaction can also be carried out on the iodo analogs of compound 2 andin some cases on the chloro analogs of compound 2.

Compound 4 is obtained as a mixture of isomers that can, but need not beseparated. Each is easily dehydrated to give a mixture of theunsaturated isomers 5 and 6. This mixture shows good binding to theestrogen receptors. The sequence 4 to 7 can be carried out in a singlepot with aqueous acid. For the purpose of preparing the desired compound7, the mixtures of alkenes 5 and 6 were not separated but hydrogenatedwith H₂/Pd/C which leads to a separable mixture of 7 and 8. These wereseparated via silica gel column chromatography. There are many otherknown hydrogenation catalysts that could have been used in place ofPd/C.

Compounds 4 can also be formed from 2 and 3 without protecting thehydroxyl group of 3. In such cases two equivalents of 2 and slightlymore than 2 equivalents of BuLi was used per equivalent of compound 3.

The structures were assigned on the basis of their proton and carbon NMRspectra. The beta isomers 6 have the natural steroid stereochemistry.These compounds bind most strongly to the estrogen receptors. They showERβ to ERα selectivity that is typically greater than 5:1.

The sequence of reactions from compound 4 to 7, that is dehydration,deprotection and hydrogenation can be changed. The choice is dictated bythe properties the various intermediates and the ease of separation. Onepossible example is dehydrogenation, hydrogenation, separation of theisomers at C9 and then deprotection.

The triethylsilane induced hydrogenolysis of the C9 hydroxyl group canalso be carried out after deprotection steps that do not causedehydration. Other known methods of hydrogenolysis of the C9 hydroxylgroup in compound 4 or a deprotected version thereof, such as RaneyNickel/ethanol or via acid catalyzed NaBH₃CN can also be used.

Example 2 Synthesis of ACD Estrogenic Compounds Via Alternate Route 1

Scheme 2 depicts an alternative route for the synthesis of compounds ofFormula I in which there is a double bond at C11-C9 or C9-C8.

Example 3 Synthesis of ACD Estrogenic Compounds Via Alternate Route 2

Scheme 3 generally depicts a method for resolving the racemic compound 4into its enantiomers.

Example 4 Experimental Procedures for Synthesis of ACD EstrogenicCompounds General

All moisture sensitive reactions were carried out under nitrogenatmosphere. Anhydrous solvents were obtained as follows: THF, Et₂O,distilled from sodium and benzophenone; CH₂Cl₂ distilled from CaH₂.Analytical TLC was performed with 0.25 mm silica gel 60F plates with 254nm fluorescent indicator from Merck. Plates were visualized byultraviolet light and treatment with acidic ceric ammonium nitrate orpotassium permanganate stains followed by gently heating. Silica gel 60,(40-60 um) was purchased from Aldrich. The ¹HNMR and ¹³C NMR wasrecorded on a Bruker Avance 500, 400 and 300 spectometer. Massspectroscopy (MS), using either electron impact (EI) or chemicalionization (CI), was performed on a V. G. Micromass 7070 HS massspectrometer with an electron beam energy of 70 eV (for EI).High-resolution mass spectroscopy (HRMS) was performed on a KratosConcept-11A mass spectrometer with an electron beam of 70 eV, or a JEOLdouble focusing magnetic sector mass spectrometer JMS-AX505H.

General Procedures: Synthesis of CD Ring Moiety 3. (Hajos-ParrishKetone)

This compound was prepared in enantiomerically pure form following thevery well-known Hajos-Parrish ketone procedures. a) Hajos, Z. G.;Parrish, D. R. Org. Synth. 1984, 63, 26; b) Micheli, R. A.; Hajos, Z.G.; Cohen, N.; Parrish, D. R.; Portland, L. A.; Sciamanna, W.; Scott, M.A.; Wehrli, P. A. J. Org. Chem. 1975, 40, 675.

Protection of the Bromophenols as TBDMS Ethers 1:

The appropriate 4-bromophenol (25 mmol) and imidazole (1.25 equiv.) weredissolved in a 1:1 DMF/THF solution (15 mL). TBDMSCI (1.25 equiv.) andDMAP (trace) were added and the reaction mixture was stirred overnightat room temperature. The mixture was diluted with distilled water (75mL) and ether (75 mL) and then extracted with ethyl acetate (3×75 mL).The organic extracts were combined, dried over MgSO₄, filtered, andevaporated in vacuo. The crude product was purified on a flash column.Elution with hexane afforded of the desired product as a clear colorlessoil in generally greater than 90% yield. All compounds produced by thisroute had ¹H and ¹³C NMR spectra in agreement with the desiredstructures.

Protection of the Bromophenols as MOM Ethers:

N,N-diisopropylethylamine (49.7 mmol) and chloromethyl methyl ether(49.7 mmol) were added to a solution of 4-bromophenol (24.9 mmol) in 30ml of dry dichloromethane (DCM) under nitrogen atmosphere at 0° C. Theresulting yellow mixture was stirred for 30 min. at 0° C. then left overnight at room temperature. The organic mixture was diluted with aq. 10%NaOH (30 ml) and extracted with dichloromethane (3×30 mL). The organiclayers were combined, dried over MgSO₄, filtered and concentrated invacuo. The crude product was purified on a silica column. Elution with15% ethylacetate in hexane afforded the desired product as yellowish oilwith yields approaching 97%. All compounds produced by this route had ¹Hand ¹³C NMR spectra in agreement with the desired structures.

Protection of the 17-OH Group in the Hajos-Parrsish Ketone as its TBDMSEther:

To a solution of the Hajos-Parrish ketone (17.85 mmol) indimethylformamide (20 mL) was added imidazole (35.7 mmol) and TBDMSCI(19.23 mmol). The reaction mixture was stirred at room temperature for 1h. The reaction mixture was diluted with EtOAc and washed with water andbrine. The organic layers were combined, dried over MgSO₄, filtered andconcentrated in vacuo. The crude yellow oil was purified on a silicacolumn. Purification of crude product afforded the desired product asclear oil in 90% yield. ¹H NMR (300 MHz, CDCl₃) δ 3.80 (t, J=4.8 Hz,1H), 2.41 (m, 2H), 2.23 (m, 3H), 1.96 (m, 2H), 1.62 (m, 3H), 1.90 (m,1H), 1.09 (s, 3H), 0.89 (s, 9H), 0.04 (s, 6H); ppm; ¹³C NMR (CDCl₃, 75MHz) δ 213.4, 79.8, 43.5, 43.4, 42.3, 36.8, 32.3, 32.1, 28.3, 25.7,20.3, 18.0, −4.5, −4.9 ppm; Mass (El) m/z 282 (0.9%, M⁺), 267 (3.8%),225 (100%); HREIMS m/z. Found for C₁₆H₃₀O₂Si: 282.2053.

Protection of the 17-OH Group in the Hajos-Parrish Ketone as its MOMEther:

To a solution of Hajos-Parrish ketone (5.95 mmol) and chloromethylmethyl ether (7.73 mmol) dissolved in DCM (15 mL) under nitrogenatmosphere was added N,N-diisopropylethylamine (7.14 mmol) at 0° C. Theresulting reaction mixture was stirred for 2 hrs at room temperature.The reaction mixture was diluted with brine (10 mL) and water (5 mL) andextracted with DCM (3×10 mL). The organic layers were combined, driedover MgSO₄, filtered and concentrated under vacuo. The crude yellowgummy product was purified on flash column, eluting with 30%ethylacetate in hexane afforded the desired product as colorless oil in75% yield. ¹H NMR (400 MHz, CDCl₃) δ 4.58 (m, 1H), 3.69 (t, J=5.7 Hz,1H), 3.30 (s, 3H), 2.43-2.28 (m, 2H), 2.34-2.07 (m, 3H), 2.05-1.82 (m,2H), 1.69-1.52 (m, 3H), 1.10 (s, 3H) ppm; ¹³C NMR (400 MHz, CDCl₃) δ212.5, 95.4, 83.9, 77.3, 77.0, 76.7, 55.1, 43.8, 42.5, 42.2, 36.5, 32.3,28.9, 28.1, 20.2 ppm.

Preparation of Enol Triflates of TBDMS Protected Hajos-Parrish Ketone:

To a cold (−78° C.) solution of lithium diisopropylamide (2.65 mmol) inTHF (10 mL) was added slowly TBDMS protected Hajos-Parrish ketone (1.77mmol) in THF (5 mL). After one hour at this temperature, a solution ofN-(phenyl)triflimide (2.12 mmol) in THF (5 mL) was introduced, and thereaction mixture was allowed to warm to room temperature for 2 h. Theresulting mixture was quenched with NH₄Cl (15 mL) and extracted withether (3×15 mL). The organic phase was dried over MgSO₄ and the solventswere evaporated under vacuo. The residue was purified by silica gelcolumn chromatography eluting with hexane to afford colorless oil (70%).¹H NMR (400 MHz, CDCl₃) δ 5.62 (m, 1H), 3.71 (m, 1H), 1.9-2.4 (m, 7H),1.2-1.6 (m, 4H), 0.92 (s, 3H), 0.86 (s, 9H), 0.01 (s, 6H); ppm; ¹³C NMR(CDCl₃, 100 MHz) δ 147.6, 122.0, 79.2, 43.2, 42.9, 39.8, 32.3, 31.1,30.0, 28.1, 27.7, 25.8, 24.6, 20.0, 19.5, −4.5, −4.9 ppm; Mass (El) m/z414.15, (100%, M⁺) HREIMS m/z. Found for C₁₇H₂₉F₃O₄SSi: 414.1508.

Coupling of a Protected A Ring Moiety with Unprotected Hajos-ParrishKetone: General Procedure A.

Protected bromophenol derivative (8.92 mmol) was dissolved in dry THF(20 mL) under nitrogen. The solution was placed in a Dry Ice/acetonebath (−78° C.) and n-butyllithium (8.92 mmol.) was added drop wise. Thesolution was stirred for 5 minutes and a solution of unprotectedHajos-Parrish ketone (2.97 mmol), dissolved in dry THF (2 mL), and addeddrop wise. The reaction mixture was quenched after 10 min with sat.NH₄Cl solution (10 mL) and of water (10 mL). The solution was extractedwith EtOAc (3×30 mL), dried over MgSO₄, filtered and evaporated undervacuo. Flash chromatography of the crude product starting the elutionwith 30% EtOAc:hexane to 50% EtOAc:hexane generally allowed one toseparate cleanly both steroisomers with overall yields approaching 80%.The isomer having the A ring in the equatorial position relative to theCD ring eluted first. All compounds produced by this route had ¹H and¹³C NMR spectra in agreement with the desired structures.

Coupling of a Protected A Ring Moiety with Either MOM or TBDMSHajos-Parrish Ketone: General Procedure B.

A suitably protected bromophenol (2.94 mmol) was dissolved in dry THF(20 mL) and placed in a Dry Ice/acetone bath (−78° C.). n-Butyllithium(2.94 mmol) was added drop wise and the solution was left to stir for 5minutes. A protected CD ring (1.67 mmol) was dissolved in dry THF (2 mL)and added drop wise. After 10 min., the reaction mixture was quenchedwith sat NH₄Cl solution (10 mL) and water (10 mL). The solution wasextracted with EtOAc (3×30 mL), dried over MgSO₄, filtered andevaporated under vacuo. The crude product was eluted with 5%EtOAc:hexane to 10% EtOAc:hexane on silica gel column afforded a mixtureof both isomers, typically in 75% yield. All compounds produced by thisroute had ¹H and ¹³C NMR spectra in agreement with the desiredstructures.

Dehydration and Deprotection of the A-CD Coupled Products. GeneralProcedure C.

A mixture of both isomers (0.146 mmol) of the condensation products of Aand CD rings obtained using one of the two general procedures A or Bdescribed above was dissolved in toluene (2 mL). A trace of paratoluenesulfonic acid (PTSA) was added. The solution was kept at roomtemperature until TLC showed the disappearance of the starting material.The mixture was concentrated in vacuo and subjected to silica gel columnchromatography. Elution with 20% ethyl acetate: 80% hexane afforded amixture of both isomers usually in more than 85% yield.

The above dehydration mixture (0.130 mmol) was dissolved in THF (3 mL)and TBAF (0.130 mmol) was added to the solution drop wise. The resultingmixture was left for 10 min, diluted with brine (1 mL) and water (1 mL)and extracted with EtOAc (3×10 mL). The organic layers were combined,dried over MgSO₄, filtered, and concentrated in vacuo. The crude productwas subjected to a silica gel column chromatography. Elution with 25%EtOAc: 75% hexane afforded the product mixture, generally in greaterthan 80% yield. All compounds produced by this route had ¹H and ¹³C NMRspectra in agreement with the desired structures.

Deprotection of the Initial A-CD Ring Coupled Products. Retaining the9-OH Group. General Procedure D.

A mixture of both isomers from condensation product of A and CD ring(0.127 mmol) produced by the general procedure A, above, in which thebromophenol was protected as its TBDMS derivative was dissolved in THF(2 mL). A THF solution of TBAF (0.127 mmol) was added, the mixture waskept for 10 minutes then diluted with brine (1 mL) and water (1 mL) andextracted with EtOAc (3×5 mL). The organic layers were combined, driedover MgSO₄, filetered, and concentrated in vacuo. The crude product wassubjected to a silica gel column eluting with 25% EtOAc: 75% hexane toafford the desired product mixture, generally in greater than 90% yield.All compounds produced by this route had ¹H and ¹³C NMR spectra inagreement with the desired structures.

Hydrogenation of the C-Ring Alkenes: General Procedure E

To a solution of unsaturated C-ring alkene ACD adduct (0.38 mmol)dissolved in methanol (5.0 mL) was added about 5-10% by weight of Pd(10% on carbon). The mixture was stirred under hydrogen atmosphere for 2h, filtered through Celite pad and washed several times with EtOAc. Thesolvent was evaporated under vacuo to afford white solid. The crude waspurified by column chromatography eluting with 45% EtOAc in hexanesafforded as a white solid with a generally more than 95% yield. In mostinstances the two isomers were separable by this procedure. In somecases the separation needed to be carried out using a preparative HPLCsystem. All compounds produced by this route had ¹H and ¹³C NMR spectrain agreement with the desired structures. Those of the key ACD compoundshaving the desired natural stereochemistry at C9 are recorded below.

Example 5 Selected Estrogenic Compounds Synthesized by Sequence andProcedures Described in Example 4

The entry numbers are used to refer to the compounds recited in thetables in the following Examples.

NMR Data for Compound, Entry 1:

¹H NMR (400 MHz, Acetone-d₆) δ 8.17 (OH), 7.07 (d, J=8.5 Hz, 2H), 6.76(d, J=8.5 Hz, 2H), 3.67 (brs, 1H), 2.65 (m, 1H), 2.21 (m, 1H), 2.09 (m,1H), 1.80 (m, 2H), 1.65 (m, 5H), 1.30 (m, 1H), 1.24 (m, 1H), 1.35 (s,3H); ppm; ¹³C NMR (Acetone-d₆, 100 MHz) δ 157.1, 140.0, 129.4, 116.7,83.2, 45.6, 43.3, 39.0, 34.2, 34.0, 33.7, 31.4, 28.2, 19.9 ppm; Mass(El) m/z 246 (48.3%, M⁺), 228 (2.5%), 202 (4.2%), 146 (10.9%), 120(100%); HREIMS m/z calculated for C₁₆H₂₂O₂246.1779.

NMR Data for Compound, Entry 2:

¹H NMR (400 MHz, Acetone-d₆): 0.90 (3H, s), 1.12-1.39 (4H, m), 1.56-1.62(4H, m), 1.72 (1H, quintet, J=6.24 Hz), 1.92 (1H, dt, J=13.73 and 3.09Hz), 2.37-2.45 (1H, m), 3.44 (1H, d, J=5.60 Hz), 4.36-4.41 (1H, m),6.84-6.91 (2H, m), 6.94-6.97 (1H, m), 8.33 (1H, s); ¹³C NMR (100 MHz,Acetone-d₆): 24.0, 28.6, 34.9, 40.9, 44.4, 44.7, 46.9, 73.9, 115.7,115.9, 119.3, 119.4, 124.4, 124.4, 141.8, 141.9, 152.0 ppm.

NMR Data for Compound, Entry 3:

¹H NMR (400 MHz, Acetone-d₆): 0.91 (3H, s), 1.13-1.20 (1H, m), 1.25-1.32(1H, m), 1.37 (1H, dd, J=13.10 and 4.62 Hz), 1.53-1.68 (4H, m), 1.74(1H, quintet, J=6.20 Hz), 1.93 (1H, dt, J=13.78 and 3.21 Hz), 2.09-2.15(1H, m), 2.69 (1H, tt, J=12.23 and 3.21 Hz), 2.89 (1H, s), 3.45 (1H, d,J=5.04 Hz), 4.36-4.40 (1H, m), 6.52 (1H, dd, J=12.26 and 2.46 Hz), 6.60(1H, dd, J=8.40 and 2.43 Hz), 7.10 (1H, t, J=8.63 Hz), 8.49 (1H, s); ¹³CNMR (100 MHz, Acetone-d₆,): 24.0, 28.6, 30.1, 34.9, 37.8, 37.9, 39.5,44.4, 46.9, 73.9, 104.2, 104.5, 113.0, 113.1, 126.3, 126.5, 129.8,129.9, 158.5, 158.6, 161.6, 164.0 ppm.

NMR Data for Compound, Entry 4:

¹H NMR (400 MHz, CDCl₃): 1.10 (3H, s), 1.24-1.28 (3H, m), 1.46-1.57 (2H,m), 1.69-1.77 (2H, m), 1.82-1.91 (1H, m), 2.06-2.13 (1H, m), 2.18-2.27(1H, m), 2.61 (1H, tt, J=12.38 and 3.69 Hz), 3.73 (1H, d, J=5.84 Hz),5.22 (1H, s), 6.76 (2H, d, J=9.00 Hz); ¹³C NMR (100 MHz, CDCl₃): 18.3,26.5, 29.2, 31.7, 32.0, 31.2, 37.4, 41.3, 44.0, 82.4, 109.7, 109.8,109.8, 109.9, 110.0, 130.6, 139.5, 150.4, 150.4, 152.8, 152.8 ppm.

NMR Data for Compound, Entry 5:

¹H NMR (400 MHz, Acetone-d₆): 1.12 (3H, s), 1.22-1.28 (1H, m), 1.35 (1H,td, J=13.24 and 3.90 Hz), 1.54-1.62 (2H, m), 1.64-1.74 (3H, m),1.76-1.84 (2H, m), 2.08-2.13 (1H, m), 2.18-2.27 (1H, m), 3.01 (2H, tt,J=12.35 and 3.84 Hz), 3.65 (1H, d, J=5.66 Hz), 6.76 (1H, td, J=8.42 and1.96 Hz), 6.95 (1H, td, J=8.19 and 2.26 Hz); ¹³C NMR (100 MHz,Acetone-d₆): 20.0, 28.2, 29.9, 32.7, 32.8, 33.9, 34.0, 43.3, 45.8, 83.2,114.4, 114.4, 123.2, 123.2, 123.2, 123.3, 128.1, 128.1, 128.2, 128.3,140.8, 143.2, 143.4, 146.0, 146.1, 146.1, 146.2, 150.0, 152.4 ppm.

NMR Data for Compound, Entry 7:

¹H NMR (400 MHz, Acetone-d₆): 1.14 (3H, s), 1.33 (2H, m), 1.69 (4H, m),1.85 (2H, m), 2.10 (1H, m), 2.26 (1H, m), 3.03 (1H, m), 3.66 (1H, d,J=5.66 Hz), 6.85 (1H, m) ppm.

NMR Data for Compound, Entry 8:

¹H NMR (400 MHz, Acetone-d6) δ 7.15 (d, J=8.8 Hz, 1H), 6.84 (d, J=2.4Hz, 1H), 6.75 (dd, J=8.4, 2.4 Hz, 1H), 4.38 (t, J=8.0 Hz, 1H), 3.48 (m,1H), 1.96-1.12 (m, 18H), 0.91 (s, 3H) ppm.

NMR Data for Compound, Entry 9:

¹H NMR (400 MHz, Acetone-d₆): 1.14 (3H, s), 1.23-1.29 (1H, m), 1.36 (1H,td, J=13.24 and 3.78 Hz), 1.48-1.54 (1H, m), 1.58-1.62 (3H, m),1.64-1.71 (1H, m) 1.80-1.87 (2H, m), 2.23 (3H, s), 2.90 (3H, s), 3.42(1H, d, J=4.25 Hz), 3.65 (1H, t, J=4.74 Hz), 6.61-6.64 (2H, m), 7.08(1H, d, J=8.34 Hz), 7.93 (1H, s); ¹³C NMR (100 MHz, Acetone-d₆): 20.2,20.4, 28.4, 33.9, 34.0, 34.6, 34.8, 43.7, 45.9, 83.3, 114.7, 118.8,128.1, 138.0, 138.0, 156.8 ppm.

NMR Data for Compounds, Entry 11:

¹H NMR (400 MHz, Acetone-d₆) δ 8.34 (OH), 7.36 (d, J=8.8 Hz, 2H), 6.88(d, J=8.8 Hz, 2H), 6.06 (m, 1H), 3.88 (m, 1H), 2.47 (m, 2H), 2.19 (m,2H), 1.89 (m, 1H), 1.71 (m, 2H), 1.45 (m, 2H), 1.09 (s, 3H); ppm; ¹³CNMR (Acetone-d₆, 100 MHz) δ 158.3, 135.6, 128.0, 121.7, 116.9, 79.6,46.4, 44.3, 34.7, 32.4, 30.3, 29.5, 26.0, 21.3 ppm; Mass (El) m/z 244(100%, M⁺), 211 (9.1%), 185 (35.5%), 146 (33.0%), 120 (44.6%); HREIMSm/z calculated for C₁₆H₂₀O₂ 244.1445.

NMR Data for Compounds, Entry 12:

¹H NMR (400 MHz, Acetone-d₆): 0.98 (3H, s), 1.29-1.37 (1H, m), 1.41-1.48(1H, m), 1.57-1.64 (1H, m), 2.10-2.18 (1H, m), 2.30-2.38 (2H, m), 2.90(2H, s), 3.52-3.59 (1H, m), 3.73-3.85 (1H, m), 5.98-6.04 (1H, m), 6.92(1H, dd, J=9.21 and 8.52 Hz), 7.07-7.10 (1H, m), 7.15 (1H, dd, J=13.09and 2.12 Hz), 8.53 (1H, s); ¹³C NMR (100 MHz, Acetone-d₆): 19.2, 19.8,23.8, 27.3, 30.2, 31.7, 32.0, 32.7, 39.8, 42.2, 42.3, 44.4, 77.6, 79.3,112.3, 112.3, 112.4, 112.5, 117.4, 117.4, 120.9, 120.9, 121.0, 121.2,127.4, 132.7, 132.7, 134.7, 134.7, 143.4, 143.6, 150.1, 152.5 ppm.

NMR Data for Compounds, Entry 13:

¹H NMR (400 MHz, Acetone-d₆): 1.00 (3H, s), 1.29-1.45 (2H, m), 1.55-1.63(2H, m), 2.30-2.35 (2H, m), 2.90 (2H, s), 3.52-3.59 (1H, m), 3.73-3.88(1H, m), 5.74-5.80 (1H, m), 6.54 (1H, dd, J=12.80 and 2.40 Hz), 6.62(1H, dd, J=8.42 and 2.45 Hz), 7.10 (1H, t, J=8.59 Hz), 8.66 (1H, s); ¹³CNMR (100 MHz, Acetone-d₆): 19.3, 20.0, 25.5, 25.5, 28.1, 31.7, 31.9,32.7, 40.0, 42.1, 44.3, 77.6, 79.3, 102.7, 102.9, 111.2, 111.2, 124.5,129.8, 129.8, 129.9, 130.6, 130.6, 131.0, 131.0, 157.5, 157.6.

NMR Data for Compounds, Entry 14:

¹H NMR (400 MHz, CDCl₃,): 1.00 (3H, s), 1.30-1.42 (1H, m), 1.45-1.67(3H, m), 2.11-2.23 (2H, m), 2.30-2.41 (2H, m), 3.81-3.90 (1H, m),5.95-6.02 (1H, m), 6.93 (2H, dd, J=1.82 and 9.92 Hz); ¹³C NMR (100 MHz,CDCl₃): 19.1, 19.8, 23.8, 27.0, 28.2, 28.9, 29.9, 31.7, 32.0, 32.5,39.5, 42.5, 44.1, 79.2, 80.5, 108.0, 108.1, 108.1, 108.2, 122.4, 128.6,131.4, 131.9, 131.9, 134.1, 150.4, 150.5, 152.8, 152.9 ppm.

NMR Data for Compounds, Entry 15:

¹H NMR (400 MHz, Acetone-d₆): 1.01-1.04 (3H, m), 1.29-1.36 (1H, m),1.38-1.46 (1H, m), 1.51-1.66 (2H, m), 2.10-2.19 (1H, m), 2.31-2.38 (2H,m), 2.92 (1H, s), 3.54-3.61 (1H, m), 3.74-3.87 (1H, m), 5.81-5.87 (1H,m), 6.73-6.78 (1H, m), 6.91 (1H, td, J=8.19 and 2.30 Hz), 9.00 (1H, s);

¹³C NMR (100 MHz, Acetone-d₆): 21.2, 21.8, 27.2, 27.2, 32.1, 33.6, 33.7,34.5, 41.8, 43.9, 44.0, 46.1, 79.5, 81.1, 114.2, 114.2, 114.3, 124.6,124.6, 124.7, 127.6, 127.6, 131.9, 133.6, 133.6, 141.0, 141.2, 143.4,146.8, 146.8, 146.9, 146.9, 149.5, 151.8 ppm.

NMR Data for Compound, Entry 16:

¹H NMR (400 MHz, Acetone-d₆) δ 7.01 (dd, J=11.6; 7.2 Hz, 1H), 6.72 (dd,J=11.6, 7.2 Hz, 1H), 5.82 (m, 1H), 3.73 (dd, J=6.4, 2.4 Hz, 1H),2.22-2.07 (m, 6H), 1.93 (dq, 1H), 1.84-1.74 (m, 2H), 1.56-1.48 (m, 1H),1.43-1.29 (m, 2H), 1.03 (s, 3H); ¹³C NMR (400 MHz, Acetone-d₆) δ 212.5,95.4, 83.9, 77.3, 77.0, 76.7, 55.1, 43.8, 42.5, 42.2, 36.5, 32.3, 28.9,28.1, 20.2 ppm.

NMR Data for Compound Entry 17:

¹H NMR (400 MHz, Acetone-d₆) δ 6.98 (dd, J=11.6; 7.2 Hz, 1H), 6.63 (dd,J=11.6, 7.6 Hz, 1H), 5.83 (dd, J=2.0, 2.0 Hz, 1H), 3.81 (t, J=6.0 Hz,1H), 2.37-2.22 (m, 3H), 2.13-1.97 (m, 7H), 1.59-1.51 (m, 2H), 1.41-1.27(m, 2H), 0.96 (s, 3H); ¹³C NMR (400 MHz, Acetone-d₆) δ 131.7, 115.6,115.5, 115.4, 115.3, 115.2, 105.2, 104.9, 77.5, 44.3, 25.3, 25.2, 19.3ppm.

NMR Data for Compounds, Entry 18:

¹H NMR (400 MHz, Acetone-d₆): 0.99 (3H, s), 1.28-1.89 (6H, m), 2.10-2.34(6H, m), 3.83 (1H, m), 5.86 (1H, m), 6.78 (1H, m) ppm.

NMR Data for Compound Entry 19:

¹H NMR (400 MHz, Acetone-d6) δ 7.01 (d, J=8.4 Hz, 1H), 6.84 (d, J=2.4Hz, 1H), 6.74 (dd, J=8.4, 2.4 Hz, 1H), 5.50 (m, 1H), 3.75 (dd, J=6.0,1.6 Hz, 1H), 2.39 (m, 1H), 2.23-2.03 (m, 5H), 1.88 (dq, 1H), 1.84-1.77(m, 1H), 1.75-1.69 (m, 1H), 1.56-1.44 (m, 2H), 1.07 (s, 3H) ppm;

¹³C NMR (Acetone-d6, 100 MHz) δ 157.0, 135.0, 134.0, 132.3, 130.8,124.7, 116.0, 114.1, 79.3, 42.1, 39.8, 32.2, 31.8, 29.7, 29.6, 29.4,29.3, 29.2, 29.0, 28.8, 28.6, 28.4, 28.0, 19.8 ppm.

NMR Data for Compound Entry 20:

¹H NMR (400 MHz, Acetone-d6) δ 7.02 (d, J=8.4 Hz, 1H), 6.85 (d, J=2.4Hz, 1H), 6.74 (dd, J=8.4, 2.4 Hz, 1H), 5.55 (m, 1H), 3.88 (t, J=6.0 Hz,1H), 2.33-2.02 (m, 7H), 1.65-1.57 (m, 2H), 1.44-1.29 (m, 2H), 1.02 (s,3H) ppm; ¹³C NMR (400 MHz, Acetone-d6) δ 156.8, 134.8, 134.3, 132.3,131.0, 130.8, 115.9, 114.1, 77.5, 44.1, 42.2, 31.9, 30.2, 29.7, 29.5,29.4, 29.2, 29.0, 28.8, 28.7, 28.6, 28.4, 26.3, 19.4 ppm.

NMR Data for Compound, Entry 21:

¹H NMR (400 MHz, CDCl₃): 1.00 (3H, s), 1.17-1.25 (1H, m), 1.34-1.42 (2H,m), 2.11-2.17 (2H, m), 2.25 (4H, m), 2.37-2.40 (2H, m), 3.79-3.90 (1H,m), 4.65 (1H, s), 5.89-5.97 (1H, m), 6.72 (1H, d, J=8.19 Hz), 7.11 (1H,dt, J=8.10 and 2.26 Hz), 7.17 (1H, m) ppm.

NMR Data for Compounds Entry 22:

¹H NMR (400 MHz, CDCl₃): 1.04-1.09 (3H, m), 1.33-1.47 (2H, m), 1.49-1.73(4H, m), 1.77-1.90 (1H, m), 2.05-2.17 (3H, m), 2.30-2.37 (1H, m),3.82-3.95 (1H, m), 5.41-5.50 (1H, m), 6.60 (1H, dd, J=8.15 and 2.60 Hz),6.64 (1H, d, J=2.55 Hz), 6.91 (1H, dd, J=8.14 and 3.22 Hz); ¹³C NMR (100MHz, CDCl₃): 19.4, 19.8, 19.9, 27.1, 28.3, 29.1, 30.2, 30.3, 31.9, 32.2,39.7, 42.2, 42.4, 43.9, 79.3, 80.7, 112.3, 116.7, 123.1, 129.4, 129.5,129.6, 135.8, 135.9, 136.7, 136.7, 136.8, 154.1 ppm.

NMR Data for Compound, Entry 24:

¹H NMR (CDCl₃, 400 MHz); δ 7.00 (dd, apparent t, J=2.8, 2.8 Hz, 1H),6.96 (dd, J=8.0, 1.2, 1H), 6.83-6.75 (m, 1H), 6.72-6.69 (m, 1H), 5.61(dd, J=17.6, 1.2 Hz, 1H), 5.46-5.44 (m, 1H), 5.20 (dd, J=10.8, 1.2 Hz,1H), 3.94 (dd, apparent t, J=5.6, 5.6, 0.4H), 3.85 (dd, J=6.4, 1.6 Hz,0.6H), 2.39-2.08 (m, 4H), 1.91-1.83 (m, 1H), 1.81-1.19 (m, 7H), 1.09 (s,1.8H), 1.05 (s, 1.2H); ¹³C NMR (CDCl₃, 100 MHz): δ 154.42, 136.60,136.57, 136.12, 135.97, 135.40, 135.32, 134.85, 134.78, 130.88, 129.80,129.73, 124.50, 114.72, 114.68, 114.20, 114.15, 111.68, 111.54, 80.68,79.29, 43.96, 42.36, 42.18, 39.76, 32.21, 32.10, 31.84, 30.77, 29.02,28.23, 27.47, 19.94, 19.38 ppm.

NMR Data for Compound, Entry 27:

¹H NMR (400 MHz, CD₃OD) δ 8.34 (OH), 7.36 (d, J=8.8 Hz, 2H), 6.88 (d,J=8.8 Hz, 2H), 3.88 (m, 1H), 2.19 (m, 2H), 1.89 (m, 1H), 1.71 (m, 2H),1.45 (m, 2H), 1.09 (s, 3H); ppm; ¹³C NMR (100 MHz, CD₃OD) δ 158.3,135.6, 128.0, 121.7, 79.6, 72.3, 44.3, 34.7, 32.4, 30.3, 29.5, 26.0,21.3 ppm.

NMR Data for Compound, Entry 28:

¹H NMR (400 MHz, Acetone-d₆): 1.02 (3H, s), 1.06-1.12 (1H, m), 1.48-1.54(1H, m), 1.63-1.72 (2H, m), 1.75-1.85 (2H, m), 1.87-1.97 (2H, m),2.09-2.21 (2H, m), 3.43 (1H, d, J=4.72 Hz), 3.64 (1H, s), 3.85-3.88 (1H,m), 6.92 (1H, t, J=8.55 Hz), 7.15 (1H, ddd, J=8.45, 2.24, and 0.92 Hz),7.26 (1H, dd, J=13.22 and 2.23 Hz), 8.46 (1H, s); ¹³C NMR (100 MHz,Acetone-d₆): 20.9, 29.5, 33.5, 36.1, 40.2, 43.6, 45.1, 74.4, 80.9,114.8, 115.0, 118.9, 118.9, 123.0, 123.0, 144.3, 144.3, 144.7, 144.9,151.7, 154.1 ppm.

NMR Data for Compound, Entry 29:

¹H NMR (400 MHz, CD₃OD): 1.00 (3H, s), 1.06 (1H, dt, J=13.85 and 4.66Hz), 1.45-1.61 (3H, m), 1.68-1.75 (1H, m), 1.79-1.85 (2H, m), 2.01-2.07(1H, m), 2.14-2.25 (2H, m), 2.29-2.36 (1H, m), 3.27-3.28 (2H, m), 3.32(1H, s), 3.81 (1H, dd, J=6.64 and 3.72 Hz), 6.42 (1H, dd, J=14.15 and2.41 Hz), 6.52 (1H, dd, J=8.60 and 2.40 Hz), 7.32 (1H, t, J=9.63 Hz);¹³C NMR (100 MHz, CD₃OD): 19.7, 29.1, 29.9, 32.5, 33.7, 33.8, 37.4,37.5, 43.0, 44.5, 74.0, 74.1, 81.0, 104.3, 104.6, 111.7, 111.7, 127.3,127.4, 129.2, 129.3, 159.0, 159.2, 160.9, 163.3 ppm.

NMR Data for Compound, Entry 30:

¹H NMR (400 MHz, CD₃OD): 0.95 (3H, s), 1.06-1.13 (1H, m), 1.21 (1H, t,J=7.14 Hz), 1.46-1.55 (1H, m), 1.60-1.76 (3H, m), 1.81-2.04 (5H, m),2.13-2.22 (1H, m), 3.29 (2H, quintet, J=1.61 Hz), 3.90 (1H, dd, J=7.04and 4.54 Hz), 6.99 (2H, d, J=10.21 Hz); ¹³C NMR (100 MHz, CD₃OD): 18.8,27.2, 28.8, 30.6, 33.5, 37.8, 41.7, 42.9, 72.5, 78.5, 108.1, 108.1,108.2, 108.3, 108.4, 104.4, 139.4, 151.1, 151.1, 153.4, 153.5 ppm.

Example 6 Oxidation of 17-OH to Ketone and its Ethynyl Derivative(Entry10)

To a solution of saturated final compound (0.2 mmol) in acetone (5 mL)was added Jones' reagent (0.22 mmol). The reaction was stirred at roomtemperature for 5 minutes. Isopropanol (0.5 mL) was added, solvent wasevaporated under vacuo, and extracted with EtOAc (3×5 mL). The organiclayers were combined, dried over MgSO₄, and evaporated under vacuo togive white gummy oil. Purification of crude product by flashchromatography afforded white solid with 90% yield. ¹H NMR (400 MHz,Acetone-d₆) δ 8.08 (OH), 7.10 (d, J=8.5 Hz, 2H), 6.76 (d, J=8.5 Hz, 2H),2.74 (m, 1H), 2.43 (m, 1H), 2.12 (m, 3H), 1.82 (m, 3H), 1.59 (m, 3H),1.17 (m, 1H), 1.11 (s, 3H); ppm; ¹³C NMR (Acetone-d₆, 100 MHz) δ 207.1,157.4, 139.6, 129.5, 116.9, 47.9, 45.0, 39.0, 37.5, 34.5, 30.4, 24.6,20.2 ppm.

A solution of the above oxidized product (1.0 mmol) in dry dimethylsulfoxide (10 mL) under nitrogen was treated with lithiumacetylide-ethylenediamine complex (300 mg), and the mixture was stirredat room temperature for 16-20 h. The mixture was poured into cold water,acidified with dilute acetic acid, extracted with ethyl acetate, washedwith water and brine, and dried over MgSO₄. After evaporation of thesolvent under reduced pressure, the residue was chromatographed onsilica gel with 10-20% ethyl acetate in hexane to yield the desiredcompound. ¹H NMR (400 MHz, Acetone-d₆) δ 7.08 (d, J=8.4 Hz, 2H), 6.74(d, J=8.4 Hz, 2H), 2.64 (m, 1H), 2.04 (m, 1H), 1.40-1.80 (m, 10H), 1.21(s, 3H); ppm; ¹³C NMR (Acetone-d₆, 100 MHz) δ 155.4, 138.2, 127.7,115.0, 88.3, 80.4, 73.0, 44.8, 42.1, 37.6, 37.3, 33.2, 29.8, 28.3, 23.1,18.8 ppm.

Example 7 Synthesis of Unsaturated Compounds

Coupling of A Ring with Hajos-Parrish Derived Enol Triflate:

To a solution of enol triflate (3.38 mmol) and (dba)₃Pd₂.CHCl₃ (0.1mmol) in DCM (18 mL) at −78° C. was added a solution of4-hydroxyphenylboronic acid (3.37 mmol) in THF (30 mL) followed byaddition of triethylamine (10 mmol). The reaction mixture was brought toroom temperature and refluxed for 1 h at 80° C., concentrated andpurified by flash chromatography on silica gel (10% EtOAc in hexanes) togive the compounds 11 as a colorless oil with 74% yield. ¹H NMR (400MHz, CDCl₃) δ 7.31 (d, J=9.0 Hz, 2H), 6.83 (d, J=9.0 Hz, 2H), 5.63 (m,1H), 3.71 (m, 1H), 1.74-2.48 (m, 6H), 1.28-1.34 (m, 3H), 0.94 (s, 3H),0.87 (s, 9H), 0.01 (s, 6H); ppm; ¹³C NMR (CDCl₃, 100 MHz) δ 158.4,147.6, 127.4, 116.0, 79.2, 42.9, 39.8, 32.1, 31.1, 27.9, 25.8, 19.9,−4.5, −4.9 ppm.

Preparation of 5-vinyl-4-bromo-phenol

PPh₃MeBr (41.1 mmol) was dissolved in dry THF (120 ml) under nitrogenatmosphere for 15 min. at 0° C. A 1.0 M solution of NaHMDS (31.5 mmol)was added dropwise and the reaction mixture was stirred for 30 min at 0°C. The 4-bromo-5-benzaldehyde (24.2 mmol) in dry THF (10 ml) was addeddrop wise to the reaction mixture. The reaction mixture was stirred for2 hours at room temperature and diluted with NH₄Cl (30 ml), andextracted with ether (3×30 mL). The organic layers were combined, driedover MgSO₄, filtered, and evaporated under vacuo. The crude product waspurified by using silica gel to afford a pure compound as yellow oilwith 91% yield. ¹H NMR (CDCl₃, 400 MHz): δ 7.44 (d, J=8.8 Hz, 1H), 7.24(d, J=3.2, 1H), 7.02 (dd, J=17.6, 11.2, 1H), 6.85 (dd, J=8.8, 3.2 Hz,1H), 5.71 (dd, J=17.6, 1.2, 1H), 5.37 (dd, J=10.8, 0.8 Hz, 1H), 5.17 (s,2H), 3.48 (s, 3H) ppm; ¹³C NMR (CDCl₃, 100 MHz): δ 156.63, 138.24,135.61, 133.41, 117.31, 116.83, 115.54, 114.37, 94.47, 55.98 ppm.

Reaction of Compound Entry 24 with Benzylmercaptan:

The benzylmercaptan (0.45 mmol) was added to compound entry 24 (0.301mmol) in CDCl₃ (2.0 mL). The reaction mixture was exposed to the lightfor two weeks. The reaction mixture was evaporated under vacuo andsubjected to flash chromatography to give the desired product in 69%yield. ¹H NMR (CDCl₃, 400 MHz); δ 7.34-7.19 (m, 6H), 6.81-6.78 (m, 2H),5.11 (s, 2H), 4.25 (dd, J=8.0, 8.0 Hz, 1H), 3.68 (s, 2H), 3.44 (s, 3H),3.31-3.23 (m, 1H), 3.20-3.13 (m, 1H), 2.69 (dd, J=8.0 Hz, 2H), 2.20-1.04(m, 19H), 0.95 (s, 3H), 0.91-0.82 (m, 1H) ppm; ¹³C NMR (CDCl₃, 100 MHz):δ 155.93, 141.43, 139.57, 138.46, 128.87, 128.39, 126.86, 126.35,119.54, 113.21, 94.26, 73.99, 73.64, 55.93, 42.56, 41.86, 40.67, 36.45,34.20, 33.72, 33.60, 30.12, 28.17, 26.46, 21.54 ppm.

Preparation of Compounds Entry 25 and 26:

The adduct described above (0.206 mmol) was dissolved in THF (1.8 mL)and water (0.2 mL). A few crystals of para-toluene sulfonic acid wereadded and the reaction mixture was refluxed for 24 hours. The reactionmixture was diluted with sat. NaHCO₃ (10 mL) and extracted withdichloromethane (2×10 mL). The organic extracts were combined, driedover MgSO₄, filtered, and concentrated under vacuo. The crude productwas purified using column chromatography afforded the de-protectedmixture of isomers as yellow solid with 79% yield. These twostereoisomers were separated by using preparative recycling HPLCequipped with reverse phase column (250×21.2 mm, 10μm) either using 40%ACN in water or 50% ACN in water. After giving either 4 or 5 recycles toget the pure isomer with 19% and 36% yield respectively.

Isomer 25;

¹H NMR (CDCl₃, 400 MHz); δ 7.33-7.28 (m, 4H), 7.26-7.21 (m, 1H), 6.89(d, J=8.0 Hz, 1H), 6.62 (dd, J=8.0, 2.4 Hz, 1H), 6.58 (d, J=2.8 Hz),5.38-5.37 (m, 1H), 4.98 (br, 1H), 3.81 (dd, J=6.8, 1.6 Hz, 1H), 3.71 (s,2H), 2.80-2.76 (m, 2H), 2.62-2.58 (m, 2H), 2.33-2.22 (m, 2H), 2.09-2.00(m, 2H), 1.89-1.69 (m, 4H), 1.60-1.54 (m, 1H), 1.51-1.41 (m, 2H), 1.07(s, 3H) ppm; ¹³C NMR (CDCl₃, 100 MHz): δ 154.19, 139.23, 138.40, 136.67,136.65, 135.35, 130.04, 128.81, 128.48, 126.96, 123.58, 115.67, 113.08,80.57, 42.15, 39.61, 36.55, 33.21, 32.75, 32.05, 31.90, 31.17, 28.27,19.90 ppm.

Isomer 26;

¹H NMR (CDCl₃, 400 MHz); δ 7.33 (m, 4H), 7.26-7.21 (m, 1H), 6.89 (d,J=8.0 Hz), 6.62 (dd, J=8.0, 2.8 Hz, 1H), 6.59 (d, J=2.4 Hz, 1H),5.46-5.45 (m, 1H), 5.21 (brs, 1H), 3.91 (dd J=5.2, 5.2, 1H), 3.71 (s,2H), 2.80-2.71 (m, 2H), 2.62-2.56 (m, 2H), 2.33-2.28 (m, 1H), 2.25-2.04(m, 4H), 1.66-1.51 (m, 3H), 1.45-1.30 (m, 2H), 1.03 (s, 3H) ppm; ¹³C NMR(CDCl₃, 100 MHz): δ 154.32, 139.13, 138.38, 136.49, 135.20, 130.00,129.88, 128.80, 128.50, 126.95, 115.75, 113.09, 79.13, 43.82, 42.34,36.57, 33.36, 32.74, 32.10, 30.18, 28.99, 21.91, 19.38 ppm.

Example 8 Comparison of CH₃ vs F at Position 5

Replacement of the 5-H by either CH₃ or F leads to rather comparableresults. In the natural saturated series the fluoro compounds bindsbetter by a factor of about 2.5; there is almost no difference in theunsaturated series.

Interestingly, the β/α ratio in both series is virtually identical. Thissuggests that either a methyl group or a fluorine substituent can beused in this position in combination with a substituent at C4 andpossibly also at C2.

Example 9 Relative Binding Assays (RBAs)

1. Ring C Saturated Compounds Having the Natural Stereochemistry at C9

Relative binding affinities (RBAs) of a variety of ring A analogs weredetermined using the method of Kuiper [31]. The RBAs are given withreference to estradiol=100 for both the estrogen alpha and estrogen betareceptor.

Entry R₁ R₂ R₄ R₅ R₁₇ RBAα RBAβ RBAβ/RBAα Estradiol 100   100   1   1. HH H H H 1.5 21.5  14.6   2. H H F H H 1.0 8.7 8.7  3. H H H F H 27.3 135.5  5.0  4. H F F H H 0.4  0.28 7.0  5. H H F F H 4.6 42.8  9.3  6. HF H F H — — —  7. H F F F H  0.19  1.73 9.1  8. H H H Cl H 55.3  168  3.2  9. H H H CH₃ H 2.8 33.6  11.9  10. H H H H C₂H  0.09  0.11 1.2

Substituents in the 4 position consistently lowered the bindingaffinity. This was not unexpected since the both ER receptors havelittle space to accommodate substituents at this position. Thus for thesmall F-atom at C4 (entry 2) in comparison to an H at C4 (entry 1), theRBA for ERβ decreased from 21.5% to 8% while the RBA for ERα droppedfrom 1.7% to 1.0%. This loss in binding affinity by a factor of 2-3appeared to be amplified in the hormonal potency, where now entry 2 wasfound to be a weak agonist for ERβ. Fluorine substituents at bothpositions 2 and 4, as in entry 7, resulted in a further reduction inbinding by a factor of about 10 (in comparison with entry 3). The RBAsfor the 4-methyl derivative were not measured since those for theprecursor unsaturated compounds, entry 21, were already very low [0.5and 0.02 for ERα and ERβ, respectively].

The docking studies showed that there was some room around C5, which inestradiol connected the saturated B-ring. The RBA studies confirmed thatsubstituents at C5 led to a very high level of activity; i.e., thisposition is strongly activating. Three derivatives of this type havebeen synthesized and assayed, entries 3, 8 and 9. The RBA was higherthan that found for the parent compound entry 1 irrespective of the typeof substituent and, in some cases, exceeded that of estradiol itself forthe ERβ receptor (entries 3 and 8, with F or Cl substituents).Interestingly, an increase in the ERβ binding affinity beyond that ofestradiol is accompanied by a disproportionately larger increase in ERαand thus a substantially lower binding selectivity. The availabletranscription activation data followed the same trend.

2. Structures with a Double Bond in Ring C Either C9-C11 or C9-C8

The compounds were tested as mixtures except as noted below for entries16, 17, 19, 20, 25 and 26 in which the separated compounds were tested(as noted)

Entry R₁ R₂ R₄ R₅ RBAα RBAβ RBAβ/RBAα Estradiol 100 100 1 11. H H H H0.21 2.0 9.5 12. H H F H 0.04 0.84 21 13. H H H F 4.5 49 10.9 14. H F FH 0.012 0.020 1.7 15. H H F F 0.78 6.8 8.7 16. (5) H F H F 0.44 2.6 5.117. (6) H F H F 0.38 3.3 6.8 18. H F F F 0.19 1.73 9.1 19.(5) H H H Cl60 118 2.0 20.(6) H H H Cl 195 331 1.7 21. H H CH₃ H 0.46 0.022 0.5 22.H H H CH₃ 7.7 52.8 6.9 23. CH₃ H H CH₃ 0.5 1.1 2.2 24. H H H CH═CH₂ 0.243.3 13.8 25.(5) H H H CH₂CH₂SCH₂Ph 0.32 0.76 2.2 26.(6) H H HCH₂CH₂SCH₂Ph 0.31 0.27 0.86

3. ACD Compounds with a 9-Hydroxy Substituent and Having the NaturalStereochemistry at C9.

Most of these compounds have low binding relative to estradiol; however,the compound with a 5-F ring A substituent binds significantly and showsstrong beta selectivity. 9-Hydroxy compounds with the un-naturalconformation generally have lower binding affinities than those havingthe natural stereochemistry.

Entry R₁ R₂ R₄ R₅ RBAα RBAβ RBAβ/RBAα 27. H H H H 0.01 0.1 10 28. H F HH 0.009 0.12 13 29. H H H F 0.17 3.6 21 30. H F F H 0.005 0.023 4.6

4. Ring C Saturated Compounds with the Non-Natural Stereochemistry atC9.

Several analogs have been tested. In most cases the binding affinityrelative to the compound with the natural stereochemistry at thisposition is quite low. Several examples are shown to illustrate thispoint. These compounds appear to be of limited value as estrogenreceptor agonists or antagonists.

Example 10 Comparison of Three 5-F Analogs

A substantial decrease [37-fold] in the binding affinity was observed ingoing from the saturated compound, Entry 3 to the 9-hydroxy compound,Entry 29. It should be noted however that the binding activity of this9-hydroxy compound is still substantial and that the beta selectivity isas high as of any of the ACD compounds tested thus far. Based on theseresults, the analagous 5-chloro-9-hydroxy compound is expected to showbinding affinity and selectivity comparable to or better than the5-fluoro analog.

Similar comparisons of the other available 9-hydroxy derivatives showedbinding decreases ranging up to 200 fold compared to the saturatedanalogs.

Example 11 Relative Transcription Assays

The relative transcription activities (RTAs) for a variety of ringanalogs were determined [35]. The RTAs are provided below with referenceto estradiol=100 for both the estrogen alpha and estrogen beta receptor.Estradiol shows essentially no selectivity for these two receptors.

Ratio RTAβ/ Entry R1 R2 R4 R5 RTAα RTAβ RTAα Estra- 100 100 1 diol  1. HH H H 4.3 164 38  1* H H H H [unnatural −8.9 7.7 NA isomer]  2. H H F H−8.3 14 NA  3. H H H F 44 162 3.6  5. H H F F 18 151 8.4  7. H F F F 1153 4.8  8. H H H Cl 88 188 2.1  9. H H H CH₃ −9.7 149 NA 19. H H H Cl120 157 1.6 20. H H H Cl 98 188 2.1 26. H H H CH₂CH₂SCH₂Ph 6.7 15.6 2.2

The transcriptional activation of the 5-F derivative, entry 3, showsthat this compound, and presumably also the 5-Cl compound, is both anERα and an ERβ agonist. This behaviour is in contrast to that of thetypical SERMs reported in the literature, which are generally eitheralpha or beta agonists, but not both. The conversion of meta-halogenatedphenols into catechols and eventually ortho-quinones has been studiedfrom using computer modelling calculations and have shown that that theformation of these carcinogenic intermediates is significantly slowerfor the halogenated compounds than from 4-methylphenol itself. Thusthese compounds have considerable potential in the field of hormonereplacement therapy.

Example 12 Quinone Related Toxicity Assays

The toxicity of selected compounds on intact hepatocytes were measuredas LC50 values after 2 hour exposure. The LC50 values represent theconcentration (in micromolar units) which caused the death of 50% of thehepatocytes in the sample population. In the present case, the toxicitylevels are thought to be correlated with the amount of quinone formed(P. O'Brien, personal communication). The results are shown below. Thedata confirms that the fully substituted phenol of compound 7 results indecrease of toxicity, perhaps to a baseline level. Compound 7 is the2,4,5-trifluoro A-CD system, where both ortho positions have beenblocked. Of the compounds tested, it shows the lowest toxicity (LC50>600micromolar). These measurements were conducted at Prof. Peter O'Brien'slab at the Department of Pharmacology, University of Toronto.

Hepatotoxicity of selected A-CD compounds Entry LC50 μM Estradiol400-450 1 320-400 2 400-450 3 250-280 5 155-200 7 >600

These data suggest that in order to prevent ortho-hyroxylation andsubsequent quinone formation, blocking of the ortho-positions, forexample, using F atoms, will form an important part of the designstrategy. Compounds whose toxicities approach the high micromolar ormillimolar range should be useful as drugs, since the dosage should bein the sub-micromolar range, thus affording a considerable safety factorin usage.

All publications, patents and patent applications mentioned in thisSpecification are indicative of the level of skill of those skilled inthe art to which the presently disclosed and claimed inventiveconcept(s) pertains and are herein incorporated by reference to the sameextent as if each individual publication, patent, or patent applicationswas specifically and individually indicated to be incorporated byreference.

The presently disclosed and claimed inventive concept(s) being thusdescribed, it will be obvious that the same may be varied in many ways.Such variations are not to be regarded as a departure from the spiritand scope of the presently disclosed and claimed inventive concept(s),and all such modifications as would be obvious to one skilled in the artare intended to be included within the scope of the following claims.

REFERENCES

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What is claimed is:
 1. A method for hormone replacement therapy in asubject, comprising administering to said subject an effective amount ofa compound of Formula I, or a pharmaceutically acceptable salt, ester orsolvate thereof:

where R₁ is H, halogen or CH₃; R₂ is H, halogen or CH₃; R₄ is H, halogenor CH₃; R₅ is H, halogen CF₃, C₁-C₅ alkyl, CH₂OH, CH₂OAc, CH₂CH₂OH,CH₂CH₂OAc, CH₂-aryl, CH₂-heteroaryl, CH═CH₂, CH₂CH₂SCH₃, CH₂CH₂SC₂H₅,CH₂CH₂SCH₂Ar, CH₂CH₂SCH₂-heteroaryl, OH, OCH₃, OC₂H₅, OCH₂Ar,OCH₂-heteroaryl, OAc, SCH₃, SC₂H₅, SCH₂Ar, SCH₂-heteroaryl, SOCH₃,SOC₂H₅, SOCH₂Ar, SOCH₂-heteroaryl, SO₂CH₃, SO₂C₂H₅, SO₂CH₂Ar,SO₂CH₂-heteroaryl, CN, CHO, COCH₃, COC₂H₅, CO₂H, CO₂CH₃, CO₂C₂H₅,CO₂CH₂Ar, CO₂CH₂-heteroaryl, CONH₂ CON(CH₃)₂, CON(CH₂)₄, CON(CH₂)₅, orNO₂; R₉ is absent, H or OH; and R₁₇ is H or C₂H.
 2. The method of claim1, wherein the compound of Formula I is further defined as having thestructure of Formula Ia:

where R₉ is H or OH.
 3. The method of claim 1, wherein, in the compoundof Formula I: R₁ is H, F or CH₃; R₂ is H or F; R₄ is H or F; R₅ is H, F,Cl, CF₃, CH₃, C₂H₅, nC₃H₇, iC₃H₇, CH₂OH, CH₂OAc, CH₂CH₂OH, CH₂CH₂OAc,CH₂-aryl, CH₂-heteroaryl, CH═CH₂, CH₂CH₂SCH₃, CH₂CH₂SC₂H₅, CH₂CH₂SCH₂Ar,CH₂CH₂SCH₂-heteroaryl, OH, OCH₃, OC₂H₅, OCH₂Ar, OCH₂-heteroaryl, OAc,SCH₃, SC₂H₅, SCH₂Ar, SCH₂-heteroaryl, SOCH₃, SOC₂H₅, SOCH₂Ar,SOCH₂-heteroaryl, SO₂CH₃, SO₂C₂H₅, SO₂CH₂Ar, SO₂CH₂-heteroaryl, CN, CHO,COCH₃, COC₂H₅, CO₂H, CO₂CH₃, CO₂C₂H₅, CO₂CH₂Ar, CO₂CH₂-heteroaryl,COHN₂, CON(CH₃)₂, CON(CH₂)₄, CON(CH₂)₅ or NO₂; R₉=H or OH; and R₁₇ is Hor ethynyl.
 4. The method of claim 3, wherein, in the compound ofFormula I, R₂ and R₄ are H.
 5. The method of claim 3, wherein, in thecompound of Formula I, R₅ is H, F, Cl or CH₃.
 6. The method of claim 1,wherein, in the compound of Formula I, said halogen is F or Cl.
 7. Themethod of claim 1, wherein, in the compound of Formula I, at least oneof R₁, R₂, R₄, and R₅ is not H.
 8. The method of claim 3, wherein, inthe compound of Formula I, R₂, R₄ and R₅ are F, and R₁, R₉ and R₁₇ areH.
 9. The method of claim 3, wherein, in the compound of Formula I, R₁,R₂, R₄, R₅, R₉ and R₁₇ are each H.
 10. The method of claim 2, wherein,in the compound of Formula Ia, R₁, R₂, R₄, R₉ and R₁₇ are each H and R₅is F or Cl.
 11. The method of claim 2, wherein, in the compound ofFormula Ia, R₁ is H, F or CH₃; R₂ is H or F; R₄ is H or F; R₅ is H, F,Cl, CF₃, CH₃, C₂H₅, nC₃H₇, iC₃H₇, CH₂OH, CH₂OAc, CH₂CH₂OH, CH₂CH₂OAc,CH₂-aryl, CH₂-heteroaryl, CH═CH₂, CH₂CH₂SCH₃, CH₂CH₂SC₂H₅, CH₂CH₂SCH₂Ar,CH₂CH₂SCH₂-heteroaryl, OH, OCH₃, OC₂H₅, OCH₂Ar, OCH₂-heteroaryl, OAc,SCH₃, SC₂H₅, SCH₂Ar, SCH₂-heteroaryl, SOCH₃, SOC₂H₅, SOCH₂Ar,SOCH₂-heteroaryl, SO₂CH₃, SO₂C₂H₅, SO₂CH₂Ar, SO₂CH₂-heteroaryl, CN, CHO,COCH₃, COC₂H₅, CO₂H, CO₂CH₃, CO₂C₂H₅, CO₂CO₂Ar, CO₂CH₂-heteroaryl,CONH₂, CON(CH₃)₂, CON(CH₂)₄, CON(CH₂)₅ or NO₂; R₉ is H or OH; and R₁₇ isH or ethynyl.
 12. The method of claim 11, wherein, in the compound ofFormula Ia, R₂ and R₄ are H.
 13. The method of claim 11, wherein, in thecompound of Formula Ia, R₅ is H, F, Cl or CH₃.
 14. The method of claim2, wherein, in the compound of Formula Ia, said halogen is F or Cl. 15.The method of claim 2, wherein, in the compound of Formula Ia, at leastone of R₁, R₂, R₄, and R₅ is not H.
 16. The method of claim 11, wherein,in the compound of Formula Ia, R₂, R₄ and R₅ are F, and R₁, R₉ and R₁₇are H.
 17. The method of claim 11, wherein, in the compound of FormulaIa, R₁, R₂, R₄, R₅, R₉ and R₁₇ are each H.
 18. The method of claim 1,wherein said method is for estrogenic hormone therapy.
 19. A method forcontraception of a female subject, comprising administering to saidsubject an effective amount of a compound of Formula I, or apharmaceutically acceptable salt, ester or solvate thereof:

where R₁ is H, halogen or CH₃; R₂ is H, halogen or CH₃; R₄ is H, halogenor CH₃; R₅ is H, halogen CF₃, C₁-C₅ alkyl, CH₂OH, CH₂OAc, CH₂CH₂OH,CH₂CH₂OAc, CH₂-aryl, CH₂-heteroaryl, CH═CH₂, CH₂CH₂SCH₃, CH₂CH₂SC₂H₅,CH₂CH₂SCH₂Ar, CH₂CH₂SCH₂-heteroaryl, OH, OCH₃, OC₂H₅, OCH₂Ar,OCH₂-heteroaryl, OAc, SCH₃, SC₂H₅, SCH₂Ar, SCH₂-heteroaryl, SOCH₃,SOC₂H₅, SOCH₂Ar, SOCH₂-heteroaryl, SO₂CH₃, SO₂C₂H₅, SO₂CH₂Ar,SO₂CH₂-heteroaryl, CN, CHO, COCH₃, COC₂H₅, CO₂H, CO₂CH₃, CO₂C₂H₅,CO₂CH₂Ar, CO₂CH₂-heteroaryl, CONH₂ CON(CH₃)₂, CON(CH₂)₄, CON(CH₂)₅, orNO₂; R₉ is absent, H or OH; and R₁₇ is H or C₂H.
 20. A method fortreating hot flashes in a subject comprising administering to saidsubject a compound of Formula I, or a pharmaceutically acceptable salt,ester or solvate thereof:

where R₁ is H, halogen or CH₃; R₂ is H, halogen or CH₃; R₄ is H, halogenor CH₃; R₅ is H, halogen CF₃, C₁-C₅ alkyl, CH₂OH, CH₂OAc, CH₂CH₂OH,CH₂CH₂OAc, CH₂-aryl, CH₂-heteroaryl, CH═CH₂, CH₂CH₂SCH₃, CH₂CH₂SC₂H₅,CH₂CH₂SCH₂Ar, CH₂CH₂SCH₂-heteroaryl, OH, OCH₃, OC₂H₅, OCH₂Ar,OCH₂-heteroaryl, OAc, SCH₃, SC₂H₅, SCH₂Ar, SCH₂-heteroaryl, SOCH₃,SOC₂H₅, SOCH₂Ar, SOCH₂-heteroaryl, SO₂CH₃, SO₂C₂H₅, SO₂CH₂Ar,SO₂CH₂-heteroaryl, CN, CHO, COCH₃, COC₂H₅, CO₂H, CO₂CH₃, CO₂C₂H₅,CO₂CH₂Ar, CO₂CH₂-heteroaryl, CONH₂ CON(CH₃)₂, CON(CH₂)₄, CON(CH₂)₅, orNO₂; R₉ is absent, H or OH; and R₁₇ is H or C₂H.
 21. The method of claim20, wherein the compound is selected from the group consisting of: